1. Field of the Invention
The present invention relates to a method for producing the bulbs of Garlic In Vitro, more practically, a method for producing the bulbs of Garlic with saving the cost for producing them and enhancing the work efficiency and the yield by dark-culturing and/or liquid media-culturing of the tissue of garlic In Vitro.
2. Description of the Related Art
Generally, the garlic is known to be propagated by vegetative propagation through dividing the clove, since their reproductive organ had been degenerated and no seeds are formed in the plant body itself. In addition, since the ratio of obtaining the clove is very low, much more amount of clove should be used as the "seeds" in cultivation of the garlic.
Furthermore, the garlic is generally exposed to a risk of possible infection of the virus, and once a parent body of the garlic is infected by the virus, the progenies resulted from the said parent body by the vegetative propagation are grown in the infected condition thereby causing the reduced yield of garlic.
In order to produce the virus-free garlic, it has been believed as the possible way, to isolate and excise the meristem tissues from the garlic, which is the sole virus-free tissue in the plant, so that the seed garlic may be produced by propagation through the tissue culture technique. However, since there consumed the significant amount of costs in producing the virus free seed garlic in the industrial scale for the practical agriculture of the farmers, such processes could not have been carried out.
For instance, the maintenance costs of tissue-culturing room for producing the seed garlic in the industrial scale according to the conventional manner comprise, generally, about 40% of labour cost, about 10% of cost for preparing the culture media, about 50% of cost for maintenance and management of the equipments and the work costs. From the above-mentioned costs, the latest costs which correspond to the largest portion is consumed for illuminating and air-conditioning of the room, heating of media according to the process and/or disposal of the waste media.
The efficiency of the basic costs for tissue-culturing depends on the rate of propagation and the amount of production of garlic microbulbs to be implanted in the ground. Therefore, the various methods for increasing the propagation efficiency have been studied, including the propagation of callus of garlic, the propagation of tissue-cultured shoot primordia, and the propagation of the shoots. However, as a result of studying the experiments and tests reported up to now, the present inventors found that the method for propagation of the garlic by tissue culture technique is the most advantageous way for increasing the yield of microbulbs.
For instance, the conventional methods for producing the virus-free body by the tissue-culturing process of garlic, in general, are carried out by the following steps:
a) isolation and excision of the meristem tissue as the virus free tissue from the parent plant body of garlic; PA1 b) culturing the said tissue obtained from the meristem and propagation of the shoots from the said cultured tissue; and PA1 c) forming the small microbulbs in vitro from the said propagated shoots. PA1 a) isolation and excision of the virus-free tissues in the length of 0.2 to 0.3 mm obtained from the meristem tissue of parent body of garlic; PA1 b) inoculating the selected and collected tissues from the meristem tissue of garlic onto the solid-type media; PA1 c) culturing the tissues inoculated onto the solid-type media under the light condition at 25.degree. C. in the culturing room for 4 weeks; PA1 d) propagating the shoots regenerated from the cultured tissues at the propagating media for 4 weeks; PA1 e) transferring the propagated shoots into the liquid-type media with additional components of 90 g/l of sucrose and plant growth regulators and culturing them primarily for 10 days; PA1 f) transferring the primarily cultured tissues into the liquid-type media having the same composition as the media used in the step e) with additional components of 140 g/l of sucrose and plant growth regulators; PA1 g) secondarily culturing the said tissues at about 25.degree. C. and under the dark-condition in the culturing room for 6 weeks; PA1 h) harvesting the microbulbs from the virus-free garlic plants in vitro PA1 in which the steps f) and g) are carried out in the altered liquid-type MS basal media under the dark-condition with no artificial illumination.
According to the above-mentioned method, it is found that the most important factors for effecting the yield of production are to providing with a suitable conditions of the environment and media for culturing, propagation and growth of the tissue in the steps of b) and c). In other words, if for the same period, the same amounts of virus-free microbulbs are produced in the suitable media and environmental condition, the main factors for the cost are the expenses of preparing the culture media, culturing equipment, and the cost for managing the electric equipments to keep the various conditions in the culturing room.
In this regard, the material costs for producing the microbulbs and the maintenances thereof mainly consist of the costs for preparing the media and keeping the illumination and electric supply and the cost for maintaining the equipment.
Conventionally, the solid type media has been used as the said media. Among the said costs, the expense for preparing the gelling agent has the main portion of the costs. Especially, the agarose media is thought as being the most preferable gelling agent. However, the price of the said agarose media as gelling agent is high, and furthermore, since the agarose is thermo-sensitive, it may be solidified only under a predetermined temperature.
Accordingly, due to the high price of the agar, much amount of expense are needed for preparing the solid type media, and since the media should be heated to above the predetermined temperature and re-solidified after carrying out the required work during the liquid state (the work should be carried only in the liquid state), the ease and efficiency of work is not satisfactory, and there also exist the problems in the processes and expenses.
As mentioned, the reason for the gelling agents such as agarose to be used in spite of the various problems, is to prevent the plants to be cultured from exhibiting the phenomena of vitrification or caulogenesis. It is found that the agar has some constituents to inhibit such phenomena (Romberger and Tabor (1971), Wernicke and Kohlenbach (1976)).
The concentration of agarose in the media has been confirmed in many studies and reports, as being directly related to the growth of plants. Especially, semi-fluid media is believed to be relatively advantageous to be used in the step before being implanted in the soil (ground), because relative to the solid media, the nutrients readily flow in the liquid media and enhance the growth of in vitro plants, and the liquid media may be easily removed from the plant body (Von Arnold and Eriksson, 1979).
Also, Faye et al. (1986) recently reported that the shoots and the plantlets grown in the liquid media are superior to those of grown in the agarose media in an aspect of uptaking the nitrogen constituents from the media.
Furthermore, Romberger and Tabor (1971) reported that the flow of large molecules is restricted in agarose media, and the growth of plants is adversely effected by the immobilization of invertase released from the culturing plants.
Debergh et al. (1981) also reported that since the micro elements and the phosphate constituents are absorbed by the agarose matrix itself, the amounts absorbed by the culturing plants are significantly reduced. Furthermore, they also reported that the absorption of the growth regulator, the mineral ions and the organic substances are restricted as the same way (Debergh, 1982). Therefore, it is believed that the agarose has the advantageous effects and functions as the media, it also has the adverse effects to restrict the growth and development of the in vitro plant.
Also, the prior liquid-type media has a problem that the rate of vitrified tissues is increased so it has not put to practical use.
For these reasons, the present inventors have researched and developed a novel and more effective media for using in culturing the tissue of garlic.
In other aspect, it was essentially known to the ordinary person skilled in the field of art that such as about 5,000 lux of illumination should be provided with the tissues cultured in the conventional solid type media for producing the microbulbs. For such illumination, throughout the period over about 6 weeks, the significant amount of the electric power should continuously be provided with the equipments in the room. For this purpose, various and many equipments for illumination should be provided in the room. Furthermore, since the additional equipments for air conditioning in the culturing room should be installed and continuously operated in order to control the room temperature within the optimum condition, which is successively raised by the illumination equipments, etc., it cannot be avoided to increase the production cost in case of the industrial scale.
As a matter of fact, since it has been believed that the illumination of the lights is essential for tissue culture of garlic, no case to approach the removal of illumination during the culturing period (that is, dark culture) has been reported in spite of such large amount of costs.